This invention relates generally to the field of cell biology of embryonic cells and neural progenitor cells. More specifically, this invention relates to the directed differentiation of human pluripotent stem cells to form cells of the neuronal and glial lineages, using special culture conditions and selection techniques.
Repairing the central nervous system is one of the frontiers that medical science has yet to conquer. Conditions such as Alzheimer""s disease, Parkinson""s disease, epilepsy, Huntington""s disease, and stroke can have devastating consequences for those who are afflicted. Traumatic injury to the head or the spinal chord can instantly propel someone from the bounds of everyday life into the ranks of the disabled.
What makes afflictions of the nervous system so difficult to manage is the irreversibility of the damage often sustained. A central hope for these conditions is to develop cell populations that can reconstitute the neural network, and bring the functions of the nervous system back in line.
For this reason, there is a great deal of evolving interest in neural progenitor cells. Up until the present time, it was generally thought that multipotent neural progenitor cells commit early in the differentiation pathway to either neural restricted cells or glial restricted cells. These in turn are thought to give rise to mature neurons, or to mature astrocytes and oligodendrocytes. Multipotent neural progenitor cells in the neural crest also differentiate to neurons, smooth muscle, and Schwann cells. It is hypothesized that various lineage-restricted precursor cells renew themselves and reside in selected sites of the central nervous system, such as the spinal chord. Cell lineage in the developing neural tube has been reviewed in the research literature by Kalyani et al. (Biochem. Cell Biol. 6:1051, 1998).
Putative multipotent neuroepithelial cells (NEP cells) have been identified in the developing spinal cord. Kalyani et al. (Dev. Biol. 186:202, 1997) reported NEP cells in the rat. Mujtaba et al. (Dev. Biol. 214:113, 1999) reported NEP cells in the mouse. Differentiation of NEP cells is thought to result in formation of restricted precursor cells having characteristic surface markers.
Putative neural restricted precursors (NRP) were characterized by Mayer-Proschel et al. (Neuron 19:773, 1997). These cells express cell-surface PS-NCAM, a polysialylated isoform of the neural cell adhesion molecule. They reportedly have the capacity to generate various types of neurons, but do not form glial cells.
Putative glial restricted precursors (GRPs) were identified by Rao et al. (Dev. Biol. 188: 48, 1997). These cells apparently have the capacity to form glial cells but not neurons.
Ling et al. (Exp. Neurol. 149:411, 1998) isolated progenitor cells from the germinal region of rat fetal mesencephalon. The cells were grown in EGF, and plated on poly-lysine coated plates, whereupon they formed neurons and glia, with occasional tyrosine hydroxylase positive (dopaminergic) cells, enhanced by including IL-1, IL-11, LIF, and GDNF in the culture medium.
Wagner et al. (Nature Biotechnol. 17:653, 1999) reported cells with a ventral mesencephalic dopaminergic phenotype induced from an immortalized multipotent neural stem cell line. The cells were transfected with a Nurr1 expression vector, and then cocultured with VM type 1 astrocytes. Over 80% of the cells obtained were claimed to have a phenotype resembling endogenous dopaminergic neurons.
Mujtaba et al. (supra) reported isolation of NRP and GRP cells from mouse embryonic stem (mES) cells. The NRPs were PS-NCAM immunoreactive, underwent self-renewal in defined medium, and differentiated into multiple neuronal phenotypes. They apparently did not form glial cells. The GRPs were A2B5-immunoreactive, and reportedly differentiated into astrocytes and oligodendrocytes, but not neurons.
A number of recent discoveries have raised expectations that embryonic cells may become a pluripotential source for cells and tissues useful in human therapy. Pluripotent cells are believed to have the capacity to differentiate into essentially all types of cells in the body (R. A. Pedersen, Scientif. Am. 280(4):68, 1999). Early work on embryonic stem cells was done using inbred mouse strains as a model (reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 6:543, 1994).
Compared with mouse ES cells, monkey and human pluripotent cells have proven to be much more fragile, and do not respond to the same culture conditions. Only recently have discoveries been made that allow primate embryonic cells to be cultured ex vivo.
Thomson et al. (Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully culture embryonic stem cells from primates, using rhesus monkeys and marmosets as a model. They subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Both hES and hEG cells have the long-sought characteristics of human pluripotent stem (hPS) cells: they are capable of ongoing proliferation in vitro without differentiating, they retain a normal karyotype, and they retain the capacity to differentiate to produce all adult cell types.
Reubinoff et al. (Nature Biotechnol. 18:399, 2000) reported somatic differentiation of human blastocysts. The cells differentiated spontaneously in culture, with no consistent pattern of structural organization. After culturing for 4-7 weeks to high density, multicellular aggregates formed above the plane of the monolayer. Different cells in the culture expressed a number of different phenotypes, including expression of xcex2-actin, desmin, and NCAM.
Spontaneous differentiation of pluripotent stem cells in culture or in teratomas generates cell populations with a highly heterogeneous mixture of phenotypes, representing a spectrum of different cell lineages. For most therapeutic purposes, it is desirable for differentiated cells to be relatively uniformxe2x80x94both in terms of the phenotypes they express, and the types of progeny they can generate.
Accordingly, there is a pressing need for technology to generate more homogeneous differentiated cell populations from pluripotent cells of human origin.
This invention provides a system for efficient production of primate cells that have differentiated from pluripotent cells into cells of the neuronal or glial lineage. Populations of cells are described which contain precursors for either lineage, which provide a source for generating additional precursor cells, the mature cells of the central nervous system: neurons, astrocytes, or oligodendrocytes. Certain embodiments of the invention have the ability to generate cells of both lineages. The precursor and mature cells of this invention can be used a number of important applications, including drug testing and therapy to restore nervous system function.
One embodiment of this invention is a cell population that proliferates in an in vitro culture, obtained by differentiating primate pluripotent stem (pPS) cells, wherein at least about 30% of the cells in the population are committed to form neuronal cells, glial cells, or both. A second embodiment is a cell population that proliferates in an in vitro culture, comprising at least about 60% neural progenitor cells, wherein at least 10% of the cells can differentiate into neuronal cells, and at least 10% of the cells can differentiate into glial cells. A third embodiment is a cell population that proliferates in an in vitro culture, comprising at least about 60% neural progenitor cells, wherein at least 10% of the cells express A2B5, and at least 10% of the cells express NCAM.
Certain cell populations of the invention are obtained by differentiating primate pluripotent stem cells, such as human embryonic stem cells. Some are obtained by differentiating stem cells in a medium containing at least two or more ligands that bind growth factor receptors. Some are obtained by differentiating pPS cells in a medium containing growth factors, sorting the differentiated cells for expression of NCAM or A2B5, and then collecting the sorted cells. Certain cell populations are enriched such that at least 70% of the cells express NCAM or A2B5.
Another embodiment of this invention is a cell population comprising mature neurons, astrocytes, oligodendrocytes, or any combination thereof, obtained by further differentiating a precursor cell population of this invention. Some such populations are obtained by culturing neural precursors in a medium containing an activator of cAMP, a neurotrophic factor, or a combination of such factors. As described below, neurons produced by such methods may be capable of exhibiting an action potential, may show gated sodium and potassium channels, and may show calcium flux when administered with neurotransmitters or their equivalents. Included are populations of cells containing a substantial proportion of dopaminergic neurons, detectable for example by staining for tyrosine hydroxylase.
Also embodied in the invention are isolated neural precursor cells, neurons, astrocytes, and oligodendrocytes, obtained by selecting a cell for the desired phenotype from one of the cell populations.
Where derived from an established line of pPS cells, the cell populations and isolated cells of this invention will typically have the same genome as the line from which they are derived. This means that the chromosomal DNA will be over 90% identical between the pPS cells and the neural cells, which can be inferred if the neural cells are obtained from the undifferentiated line through the course of normal mitotic division. Neural cells that have been treated by recombinant methods to introduce a transgene (such as TERT) or knock out an endogenous gene are still considered to have the same genome as the line from which they are derived, since all non-manipulated genetic elements are preserved.
A further embodiment of the invention is a method of screening a compound for neural cell toxicity or modulation, in which a culture is prepared containing the compound and a neural cell or cell population of this invention, and any phenotypic or metabolic change in the cell that results from contact with the compound is determined.
Yet another embodiment of the invention is a method for obtaining a polynucleotide comprising a nucleotide sequence contained in an mRNA more highly expressed in neural progenitor cells or differentiated cells, as described and exemplified further on in this disclosure. The nucleotide sequence can in turn be used to produce recombinant or synthetic polynucleotides, proteins, and antibodies for gene products enriched or suppressed in neural cells. Antibodies can also be obtained by using the cells of this invention as an immunogen or an adsorbent to identify markers enriched or suppressed in neural cells.
A further embodiment of the invention is a method of reconstituting or supplementing central nervous system (CNS) function in an individual, in which the individual is administered with an isolated cell or cell population of this invention. The isolated cells and cell populations can be used in the preparation of a medicament for use in clinical and veterinary treatment. Medicaments comprising the cells of this invention can be formulated for use in such therapeutic applications.
Other embodiments of the invention are methods for obtaining the neural precursor cells and fully differentiated cells of this invention, using the techniques outlined in this disclosure on a suitable stem cell population. Included are methods for producing cell populations containing dopaminergic cells at a frequency of 1%, 3% or 5%xe2x80x94and populations of progenitor cells capable of generating dopaminergic cells at this frequencyxe2x80x94from primate embryonic stem cells. This is particularly significant in view of the loss in dopamine neuron function that occurs in Parkinson""s disease. The compositions, methods, and techniques described in this disclosure hold considerable promise for use in diagnostic, drug screening, and therapeutic applications.
These and other embodiments of the invention will be apparent from the description that follows.